Our long-term aim is to establish real-time fiber-optics confocal microscopy (FCM) as an intraoperative imaging modality in cardiac surgery, particularly in pediatric open-heart surgery. A major risk of cardiac surgery is damage to the conduction system, which is responsible for initiating electrical signals, conducting them rapidly through the heart and synchronizing mechanical activation. Damage to the conduction system is associated with significant morbidity and (in extreme cases) necessitates costly pacemaker insertion. Despite individual variations in the conduction system, discrimination of this tissue during surgery is currently limited to the visual identification of anatomical landmarks. Our preliminary studies on several animal models and human tissue indicate that microscopic images of cardiac tissue provide sufficient information for visual discrimination of tissue of the conduction system and working myocardium. Furthermore, we identified automated methods for tissue discrimination based on quantitative texture analysis of microscopic images. In this project, we will further develop this real-time imaging modality and analysis methods so that clinicians can use the approach during surgical procedures to avoid damage to tissue of the cardiac conduction system. We will evaluate our approaches in explanted human hearts, in-vivo ovine model of neonatal hearts, and finally in the clinical setting during open-heart surgery through three Specific Aims. In Specific Aim 1, we will advance our approaches for cardiac tissue discrimination in normal and congenitally deformed human hearts. We will characterize atrial, ventricular, sinoatrial node, atrioventricular node and His bundle tissue using three-dimensional reconstructions from conventional scanning confocal microscopy. The studies will be complemented with real-time FCM imaging of these tissue. In Specific Aim 2, we will evaluate the ability of real-time FCM to guide pediatric heart surgery using an ovine model of the cardioplegic arrested neonatal heart. The tissue imaging will be performed by two different techniques of dye delivery; through the aortic root versus local surface application. Additionally, the tissue, which is identified as tissue of the conduction system will be dissected out for analysis and validation. In Specific Aim 3, we will study the microstructure of nodal tissues in-vivo in patients undergoing cardiac surgery. We will initially study this in patients undergoing a simple cardiac surgical procedure. Subsequently, we will randomize patients to FCM imaging versus standard surgical repair groups. We will investigate the impact of intraoperative FCM use on the incidence of postoperative conduction abnormalities. We hypothesize that using FCM will help decrease the incidence of postoperative conduction problems. All specific aims are designed towards testing our hypothesis that real-time FCM allows surgeons to discriminate between working myocardium and tissue of the conduction system. In summary, the proposed studies constitute a crucial step towards translation of intraoperative FCM for clinical applicatio in pediatric heart surgery.